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Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases

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Abstract

Environmental DNA from soil and water samples was extracted to construct a plasmid library and a fosmid library containing 19,500 and 20,400 clones, respectively. Two esterases (EstP2K and EstF4K) were finally isolated from each library based on activity screening, and both of them were characterized in this study. The esterase EstF4K consists of 396 amino acids with an SMTK motif which belongs to family VIII esterase/lipase. The amino acid sequence of EstF4K showed 83 % identity with that of EstA3, a reported esterase isolated from uncultured organisms of soil. EstP2K is composed of 224 amino acids in size and shows only 37 % identity with a putative lipase of Neisseria elongata subsp. The purified EstF4K was optimally active at pH 8.0 and 50 °C. It was remarkably active and very stable in the presence of 30 % dimethyl sulfoxide. Activity fingerprint of EstF4K displayed a higher level of activity toward short-chain fatty acid p-nitrophenyl (pNP) esters, while EstP2K preferred bias for pNP caprylate ester. The optimum reaction temperature and pH for EstP2K are 45 °C and 7.5, respectively, and the enzyme exhibited strong tolerance in the presence of 30 % methanol. EstF4K and EstP2K showed opposite enantioselectivity for methyl 3-phenylglycidate, a chiral synthon for the synthesis of Taxol® side chain.

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Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (no. 20773038 and 31071604), the Ministry of Science and Technology, P.R. China (no. 2011CB710800 and 2011AA02A210), and the Shanghai Municipal Fund for Natural Science (no. 07ZR14030).

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  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic of China

    Li-Ming Ouyang, Jia-Ying Liu, Ming Qiao & Jian-He Xu

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  1. Li-Ming Ouyang
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  2. Jia-Ying Liu
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  3. Ming Qiao
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  4. Jian-He Xu
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Correspondence to Li-Ming Ouyang or Jian-He Xu.

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Ouyang, LM., Liu, JY., Qiao, M. et al. Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases. Appl Biochem Biotechnol 169, 15–28 (2013). https://doi.org/10.1007/s12010-012-9949-4

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  • DOI: https://doi.org/10.1007/s12010-012-9949-4

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